Background
Parallel to the increasing global incidence of diabetes, the prevalence of diabetes in women of childbearing age is steadily rising. Diabetic pregnancy has been associated with a higher risk of adverse outcomes for the mother as well as the offspring compared to non-diabetic pregnancy [
1‐
4]. Congenital heart defects are the most common malformations observed in offspring of diabetic pregnancies, with an eightfold increase compared to non-diabetic pregnancies [
5]. Similar risks for cardiac malformation have been reported for pre-gestational type 1 or type 2
diabetes mellitus [
5]. Since type 1 and type 2 diabetes have different etiologies, these results indicate that the adverse effects on heart development are induced by pathological processes shared by both types of diabetes, such as hyperglycemia, hypoxia, oxidative stress, and abnormal maternal/fetal fuel metabolism.
In addition to the direct teratogenicity of maternal diabetes, the intrauterine and early postnatal environment can influence the cardiovascular and metabolic health of offspring later in life. This phenomenon is termed fetal or developmental programming [
6]. The offspring of a diabetic pregnancy show differences in metabolic, cardiovascular and inflammatory variables compared to the offspring of non-diabetic mothers [
7‐
9]. However, the precise mechanisms for the underlying penetrance and disease predisposition remain poorly understood.
Hypoxia plays an important role in all diabetic complications [
10‐
12], including the complications associated with diabetic pregnancy [
2,
13‐
15]. The main regulator of responses to a hypoxic environment is hypoxia-inducible factor 1 (HIF-1). HIF-1 consists of two subunits, HIF-1α, which is an O
2-labile subunit, and HIF-1β, which is constitutively expressed [
16]. HIF-1α is also important for normal embryonic development since mice with a homozygous deletion of the
Hif1a gene die due to cardiac malformations and vascular defects [
17].
Hif1a heterozygous mutants (
Hif1a+/−) normally survive past embryonic development; however,
Hif1a+/− mice demonstrate impaired responses when challenged with hypoxic conditions after birth [
18‐
20]. Cardiac myocyte-specific
Hif1a deletion causes reductions in contractility, vascularization, and also alters the expression of multiple genes in the heart during normoxia [
21]. HIF-1α is destabilized by hyperglycemia leading to the loss of cellular adaptation to low oxygen in diabetes [
10,
22]. Previously, we showed that decreased levels of
Hif1a in combination with a diabetic environment were associated with increased susceptibility to diabetic embryopathy [
23]. We found a decreased number of embryos per litter and increased incidence of heart malformations, particularly atrioventricular septal defects and reduced myocardial mass in diabetes-exposed
Hif1a+/− mice compared to
wild type (
Wt) littermates. To extend our previous study on the effects of global heterozygous deletion of
Hif1a and maternal diabetes exposure on heart development in embryos [
23], we analysed the heart of the adult offspring in the same experimental paradigm. We examined the relationship between a partial deficiency of HIF-1α and an intrauterine exposure to maternal diabetes in the fetal programming of the heart.
Discussion
In this study, we uncovered a molecular mechanism for the underlying penetrance and disease predisposition in the offspring associated with exposure to maternal diabetes. The combination of Hif1a insufficiency and exposure to diabetes in utero leads to the accelerated development of cardiac LV dysfunction. RNAseq analysis showed changes in the global expression profile of the LV of Hif1a+/− heart, indicating transcriptional reprogramming as a consequence of exposure to maternal diabetes. This reprogramming was associated with major changes in HIF-1 regulated pathways, as 53% of identified differentially expressed genes were direct and predicted HIF-1 targets. Thus, the combination of maternal diabetes and Hif1a haploinsufficiency results in significant metabolic, structural, and functional changes in the LV myocardium of the offspring.
Both human and animal studies have shown that exposure to diabetes in utero increases cardiovascular risk factors in the offspring [
5,
7,
8,
49]. In agreement with these reports, our study showed that exposure to maternal diabetes during the fetal and perinatal period compromised cardiovascular function of the adult offspring, as indicated by smaller fractional shortening. For the first time to our knowledge, the current study demonstrated that
Hif1a haploinsufficiency significantly worsens the cardiac function of the diabetes-exposed offspring. Additionally, we identified an unknown gene-environment interaction between a genetic deficiency in
Hif1a and maternal diabetes that affects the size and shape of the heart of the
Hif1a+/− offspring. The globular cardiac shape has been observed in children with fetal growth restriction and associated with systolic dysfunction as a result of the intrauterine chronic hypoxia-induced changes in cardiac development [
50].
Maternal diabetes negatively affects embryonic development and growth, and compromises placental function [
5,
13,
51]. Although here we focused on the heart of the adult offspring, due to the global nature of the
Hif1a deletion, we cannot exclude the contribution of placental dysfunction to the programmed outcome phenotype. HIF signalling, in particular, plays an important role in the development and functions of the placenta, and hypoxia-induced placental pathologies have been associated with fetal programming [
15,
52‐
54].
Our genome-scale transcriptome analyses clearly identified a set of pathophysiological processes affected by the maternal diabetes exposure and
Hif1a+/− genotype in the LV of offspring. Specifically, we found changes predominantly in metabolic processes, alterations in the innate and adaptive immune responses, apoptosis, and changes in genes associated with developmental processes. In contrast, the
Wt offspring from diabetic pregnancies show only significant enrichment of genes involved in immune system processes and inflammatory responses. Clinical studies imply that women with gestational diabetes are at a higher risk of cardiovascular diseases in association with inflammatory dysregulation [
3,
4,
55,
56]. Correspondingly, we found an increased number of infiltrating F4/80
+ macrophages in the LV of
Wt, but not in
Hif1a+/− offspring of diabetic pregnancies (Fig.
4). Altered macrophage migration in the
Hif1a+/− myocardium is consistent with a recent finding that HIF-1α has an essential role in macrophage inflammatory responses in other physiological settings [
32]. The observed increase in TUNEL staining suggests that reduced macrophage recruitment to
Hif1a+/− hearts may lead to decreased phagocytosis of apoptotic cells, which may negatively affect the maintenance of tissue integrity and lead to impaired heart function [
32].
Beside apoptosis, interstitial fibrosis is another important process of cardiac ventricular remodeling, contributing to both systolic and diastolic contractile dysfunction. Collagen secretion is stimulated by TGF-ß1 within the heart [
57]. Although increased cardiac fibrosis is a hallmark of structural remodelling in response to a diabetic environment [
58], we paradoxically found decreased collagen deposition along with decreased expression of
Tgfb1 in both
Hif1a+/− and
Wt diabetes-exposed LV at 12 weeks of age (Fig.
5). Diminished collagen deposition was also detected in the hearts of
Hif1a+/− offspring from a non-diabetic pregnancy (Fig.
5). HIF-1 regulates multiple steps in collagen biogenesis and HIF-1α knockdown results in reduced collagen deposition [
59]. Consistent with this finding,
Hif1a haploinsufficiency may affect collagen levels in the heart of
Hif1a+/− mice. Similarly, the diabetic environment destabilizes HIF-1α and alters HIF-1 regulation [
10,
22] that may affect collagen metabolism in the heart of offspring, resulting in the reduction of collagen fibrils. However, investigating the link between in utero exposure to maternal diabetes and cardiovascular outcomes in offspring is complicated by the multitude of factors that may be present at different time points. Moreover, fibrotic responses are affected by age and duration of disease, when the decreased levels of collagen may represent an intermediate stage of cardiac remodelling associated with degradation of the extracellular collagen matrix [
60]. Thus, disturbance in the synthetic and degradative aspects of collagen metabolism results in profound structural and functional abnormalities of the heart. A loss of collagen fibrils is associated with LV remodelling during volume overload and with ECM remodelling in dilated cardiomyopathy [
61]. Decreased collagen expression has been reported in children with failing single ventricle congenital heart disease [
62]. Although, based on our current data, we cannot determine whether reduced collagen content represents adverse ECM remodelling that predisposes to cardiac dysfunction, our results clearly indicate that the exposure to maternal diabetes and
Hif1a haploinsufficiency significantly affect ECM structure and composition compared with a normal myocardium.
Another mechanism implicated in the pathophysiology of the diabetic environment is enhanced production of AGEs. In the heart, protein glycation reactions alter the physiological properties of extracellular matrix proteins and cause intracellular changes in vascular and myocardial tissue [
37]. As such, AGEs represent a cardiovascular risk factor in the development of macro- and microvascular complications in diabetic patients [
63]. The increased levels of these pro-oxidant diabetogenic products correlate with increased oxidative stress, inflammation, and apoptosis in diabetic animals as well as in their progeny [
64]. We found a significant accumulation of AGEs in the LV of
Hif1a+/− offspring from a diabetic pregnancy (Fig.
6). These results provide new insights into the role of HIF-1α haploinsufficiency in susceptibility to enhanced accumulation of AGEs due to maternal diabetes exposure. Given that (i) HIF-1 regulates glucose metabolism and glycolysis to minimize oxidative stress [
16], (ii) diabetic
Hif1a+/− mice compared to diabetic
Wt have increased serum glucose and AGEs [
65], and impaired glucose homeostasis [
66], it is tempting to speculate that deficiency in HIF-1 regulation during embryonic development results in increased AGEs in the cardiac tissue of
Hif1a+/− offspring due to systemic changes in glucose metabolism and oxidative stress in the maternal diabetes environment. To clarify this question raised by our model, detailed analyses of the levels of tissue AGE modifications during embryonic and early postnatal development are now needed.
Besides structural remodelling, we identified changes in the expression of genes associated with metabolic processes that may contribute to impaired cardiac performance and increase the risk of developing heart disease in the offspring from diabetic pregnancy. Alterations in myocardial energy substrate are predominantly represented by changes in the ratio of fatty acid oxidation and glucose oxidation and have been associated with the development of cardiac pathologies [
44]. During perinatal cardiac development, the heart undergoes a switch in energy substrate preference from glucose in the fetal period to FAs. In the diabetic environment, the fetal heart is exposed to abnormal levels of substrates that may remodel cardiac metabolism of the offspring. We found significantly decreased expression of gene encoding the fatty acid translocase,
Cd36, in the heart of
Hif1a+/− offspring from a diabetic pregnancy compared to other groups (Fig.
7). Decreased myocardial levels of CD36 have been found detrimental, even in the absence of elevated circulating FAs, and to contribute to cardiac dysfunction [
67]. The decreased
Cd36 expression observed in the hearts of
Hif1a+/− offspring of diabetic pregnancies is consistent with a previous report that
Cd36 expression is regulated by HIF-1 [
45]. Expression of another direct HIF-1 target, the glycolytic gene
Ldha [
68], was significantly reduced in the LV by
Hif1a haploinsufficiency (Fig.
7). Since optimal bioenergetics are an important prerequisite for contractile efficiency, any abnormalities resulting in decreased energy production, energy transfer and energy utilization may compromise cardiac function.
To further investigate changes in angiogenesis, indicated by our RNAseq, we analysed the expression of VEGFA in the heart of the offspring. We detected a decrease in cardiac
Vegfa mRNA in the LV of both
Hif1a+/− and
Wt offspring from diabetic pregnancies (Fig.
8). Indeed, several reports have demonstrated that the expression of
Vegfa mRNA and protein are decreased in the myocardium of diabetic, insulin-resistant animals, and in diabetic patients, and have been associated with vascular abnormalities in the diabetic heart and with diabetic cardiomyopathy [
69,
70]. Cardiomyocyte-specific
Vegfa deletion demonstrates that cardiac myocytes are a major source of VEGFA in the heart and that the development and maintenance of coronary macrovasculature are compensated by non-cardiomyocyte
Vegfa expression [
71]. This phenotype also implies a different signalling mechanism for vasculogenesis/angiogenesis in the myocardium and in the coronary vasculature. In line with these data, we detected a different
Vegfa expression pattern in the cardiac tissue and in the wall of large coronary vessels. VEGFA levels in the coronary vessels were increased by the combination of
Hif1a haploinsufficiency and diabetes exposure. A similar expression profile with reduced
Vegfa levels in the cardiac tissue and increased
Vegfa expression in macrovascular tissues was reported in diabetic patients [
48]. Thus, these paradoxical changes in the expression of
Vegfa suggest that local regulatory factors differ between the myocardium and blood vessels. It is conceivable that pathophysiological conditions, such as diabetes or hypoxia, alter the regulation of
Vegfa expression in the coronary macrovasculature and myocardium.